Temporal evaluation of efficacy and quality of tissue repair upon laser‐activated sealing

Abstract Injuries caused by surgical incisions or traumatic lacerations compromise the structural and functional integrity of skin. Immediate approximation and robust repair of skin are critical to minimize occurrences of dehiscence and infection that can lead to impaired healing and further complication. Light‐activated skin sealing has emerged as an alternative to sutures, staples, and superficial adhesives, which do not integrate with tissues and are prone to scarring and infection. Here, we evaluate both shorter‐ and longer‐term efficacy of tissue repair response following laser‐activated sealing of full‐thickness skin incisions in immunocompetent mice and compare them to the efficacy seen with sutures. Laser‐activated sealants (LASEs) in which, indocyanine green was embedded within silk fibroin films, were used to form viscous pastes and applied over wound edges. A hand‐held, near‐infrared laser was applied over the incision, and conversion of the light energy to heat by the LASE facilitated rapid photothermal sealing of the wound in approximately 1 min. Tissue repair with LASEs was evaluated using functional recovery (transepidermal water loss), biomechanical recovery (tensile strength), tissue visualization (ultrasound [US] and photoacoustic imaging [PAI]), and histology, and compared with that seen in sutures. Our studies indicate that LASEs promoted earlier recovery of barrier and mechanical function of healed skin compared to suture‐closed incisions. Visualization of sealed skin using US and PAI indicated integration of the LASE with the tissue. Histological analyses of LASE‐sealed skin sections showed reduced neutrophil and increased proresolution macrophages on Days 2 and 7 postclosure of incisions, without an increase in scarring or fibrosis. Together, our studies show that simple fabrication and application methods combined with rapid sealing of wound edges with improved histological outcomes make LASE a promising alternative for management of incisional wounds and lacerations.

Together, our studies show that simple fabrication and application methods combined with rapid sealing of wound edges with improved histological outcomes make LASE a promising alternative for management of incisional wounds and lacerations.

K E Y W O R D S
incisional wounds, laser-activated sealing, photoacoustic imaging, skin barrier function recovery, tissue adhesive, tissue repair, ultrasound 1 | INTRODUCTION Soft tissue trauma, including lacerations and surgical incisions, require effective and rapid closure in order to minimize blood loss, prevent infection and promote healing. Surgical sutures and staples are the most commonly used devices for approximating soft tissue trauma including in the skin. 1,2 Although effective in superficial layers of skin, sutures do not integrate with the tissue, do not lead to immediate closure, and generally do not demonstrate optimal performance in deeper layers of the tissue, including in the hypodermis. In addition, tissue strength is suboptimal at early times after tissue approximation, which, by itself and in case of infections, can compromise effective healing.
Localized conversion of laser light energy to heat energy using endogenous or exogenous chromophores 3 results in rapid photothermal sealing of soft tissues. [3][4][5][6][7][8][9][10][11] Laser-activated sealants (LASEs) in which exogenous chromophores are incorporated within a biomaterial sealant matrix offer promising alternatives to sutures and staples. In this approach, laser irradiation of the LASE-tissue interface and the concomitant photothermal response can facilitate interdigitation of LASE biomolecules and tissue proteins, which results in rapid sealing and effective repair of soft tissues. We have previously reported the fabrication and characterization of LASEs as an approach for the rapid sealing and repair of ruptured tissues. [11][12][13][14][15] The LASE system comprises of three components: (i) a matrix consisting of biomaterials, such as elastin-like polypeptides, collagen, or silk fibroin, which integrate with the tissue upon sealing and act as a scaffold for aiding repair, (ii) chromophores including gold nanorods (GNRs) or the FDAapproved dye, indocyanine green (ICG), which convert laser light energy to heat energy (photothermal energy conversion), thus resulting in a local increase in temperature, and (iii) a hand-held nearinfrared (NIR) laser tuned to 808 nm that is used to carry out the tissue sealing procedure using LASEs. The rapid bonding of wound edges mediated by interdigitation of tissue proteins, leading to rapid sealing, has been demonstrated for temperatures ranging from 50-60 C. 16,17 A recent report which investigated the effect of temperature on tissue sealing observed highest welding strengths of tissue at 55 C. Use of elevated temperatures greater than or equal to 65 C led to denaturation of tissue proteins and negatively impacted tissue tensile strength. 18 Our previous results have shown that LASE-mediated tissue sealing results in improved recovery of tissue biomechanical properties in live mice, compared to Vetbond, a cyanoacrylate-based skin glue. 11,14 In addition to facilitating sealing and repair, LASEs can be loaded with antibacterial drugs in order to combat methicillinresistant Staphylococcus aureus infection in at surgical site, thus protecting the tissue. 15 ICG is an FDA-approved dye that absorbs and emits light in the NIR region of the wavelength spectrum. Upon irradiation with NIR lasers, approximately 85% of the energy absorbed by the dye is converted into heat, which makes ICG a good photoconverter for various applications including photothermal sealing and photodynamic therapy. 19,20 In addition, ICG dye has a relatively short clearance period of 60-80 min from the body and is excreted unchanged via bile. 21 The biodistribution and toxicity profiles of ICG dye are better understood compared to that for nanoparticles that are used as chromophores. It may also be possible to minimize batch-to-batch variation in LASE properties and performance by using the well-established ICG dye.
In this study, we carried out a detailed investigation into the efficacy of laser tissue sealing using functional, biomechanical, visual (imaging), and histological evaluation at different time points during the course of healing following surgery, and compared these outcomes to those seen with sutures. ICG dye-loaded silk fibroin ("silk") films were used for sealing 1 cm, full-thickness incisional wounds in BALB/c immunocompetent mice and transepidermal water loss (TEWL), and ultimate tensile strength (UTS) of skin were determined in order to investigate functional and biomechanical recovery, respectively, following tissue approximation. A combination of ultrasound (US) and photoacoustic imaging (PAI) along with histological evaluation was carried out in order to further visualize and gain insights into LASE-mediated tissue repair. [22][23][24][25][26][27] These findings indicate that LASEmediated tissue sealing is significantly more effective at restoring function and biomechanical properties of skin compared to sutures at early time points following surgery.

| LASE fabrication
Silk fibroin was extracted from B. mori silkworm cocoons using previously described protocols. 14

| Sealing of full-thickness incisional wounds in mice
BALB/c mice (10-12 weeks, weighing~22-25 g; Charles River Laboratories) were used in this study and were housed in groups of five until surgery. All animal care and procedures were performed in strict compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Arizona State University. Before surgery, mice were anesthetized with 120 mg/kg ketamine and 6 mg/kg xylazine (100 μl cocktail) by intraperitoneal injection. Dorsal hair was clipped, and the skin was prepped using three alternating swabs of chlorhexidine gluconate and 70% isopropyl alcohol. Two 1-cm fullthickness incisions were made side-by-side on the back of each mouse spaced roughly 2 cm apart using sterile scalpel blades (#15; Integra Miltex). 29 In case of suture-closed incisions, four evenly spaced simple interrupted knots were used to close a 1-cm incision using 4-0 nylon suture (#SN5699G; Medtronic; Monosof Black 18 00 P-13 cutting). For LASE-sealed incisions, 10 μl of phosphate-buffered saline (PBS; pH 7.4) were topically applied and a 1.2 cm Â 0.5 cm LASE film was placed over the incision; contact of LASE with PBS resulted in quick dissolution of the film to form a viscous paste between the incision edges. The incision edges were approximated using a forceps, and the incised edges were aligned prior to laser sealing. The LASE-tissue interface (incision line) was irradiated at a rate of 0.5 mm/s with the NIR laser tuned to 808 nm (CW) for 1 min while keeping the incision line approximated using forceps.
The laser was applied at an angle between 60 and 80 to the skin at a power density of~5.1 W/cm 2 (~160 mW power output, 2 mm laser beam diameter), corresponding to temperatures in the range of 50-60 C at the skinÀLASE surface. 15 Closure of left and right incisions with sutures or LASE were randomized. The mice were allowed to recover on heating pads until mobile and were housed individually. Incisions were assessed every day for up to 7 days postsurgery for any signs of infection, suture removal, or wound dehiscence and mice with any of these conditions were removed from the study.

| Measurement of TEWL of healing wounds
TEWL is a measurement of change of water vapor density across the stratum corneum layer and is used to assess the barrier function of skin. Disruption to skin due to trauma, injury, wounds results in elevated TEWL levels and is indicative of weaker barrier function. 30,31 In this study, TEWL was measured on Days 2, 4, 7 postsurgery using a portable, closed chamber VapoMeter device (#SWL5580; Delfin Technologies). The VapoMeter was fitted with a small adapter (4.5 mm diameter,~16 mm 2 surface area) and a closed chamber was created on skin contact during the duration of measurement (~9-15 s). Ambient relative humidity and temperature ( C) were recorded during every measurement using a room sensor (#RHD1367) supplied along with the VapoMeter. For every TEWL reading, three consecutive readings were acquired along nonoverlapping regions over an incision area and the chamber was passively ventilated between every measurement. 32,33 TEWL readings of unwounded skin of sham mice were acquired on the same days. In all cases, TEWL values were recorded using the DMC software (Delfin Technologies) and values are displayed as mean ± standard error of mean from six independent mice (n = 6). Spectral unmixing was performed on the obtained PA signal to discern the tissue constituents. The VevoLAB software (VisualSonics)

| Biomechanical recovery of skin strength
is utilized for such spectral unmixing, where three wavelength components (680, 800, 960 nm) are used to discern LASE signal from that of the weakly absorbing normal skin. Multi-wavelength unmixing for ICG content carried out using the Vevo system has been shown previously to produce accurate results for deep tissue imaging in tissue phantoms and murine subjects. 34,35 Control skin has a relatively weak and flat PA spectrum across the wavelength band, implying that the strong ICG absorption at 800 nm can be used to identify LASE within the samples (Figure 1c), considering that ICG is mixed with silk to form the LASE.

| Statistical analyses
Data from absorbance, TEWL, and skin UTS are presented as mean ± standard error of the mean. Differences between groups were assessed using two-way analysis of variance followed by Fisher's LSD test using GraphPad Prism version 9.2.0 (GraphPad). A p < 0.05 was considered statistically significant.

| RESULTS AND DISCUSSION
Laser sealing is an attractive approach for the sutureless approximation of tissues, including skin, and possesses several potential advantages including fast operation times, low scarring, and faster recovery of tissue function. However, the temporal dependence of the efficacy and quality this approach has not been investigated thoroughly. We, therefore, carried out detailed studies to investigate the efficacy of laser sealing in live mice in a temporal manner and compared findings with those seen with sutures. In addition to biomechanical recovery with UTS, functional recovery of barrier function of skin (using TEWL measurements), US and photoacoustic visualization and histology studies were used to develop a more comprehensive investigation into the quality and efficacy of tissue repair following laser sealing.

| Generation and characterization of LASE films
Silk fibroin ("silk")-ICG LASE films (Figure 2a) were prepared using solvent evaporation methods as described in our previous reports. 15 Briefly, aqueous solutions of silk fibroin (6 wt% or 60 mg/ml) with 0.1 mM ICG were cast and dried overnight at RT resulting in the generation of LASE films following solvent evaporation. Light absorption analyses indicated that the LASE films displayed a characteristic absorbance similar to that of ICG dye (Figure 2b). No significant shifts in absorbance profiles of these irradiated viscous LASE pastes were seen (Figure 2b). The retention of absorbance properties by laser-irradiated LASEs warrants the use of optical visualization methods for probing LASE following tissue sealing in subsequent applications.
We also investigated the photothermal response of LASE films  To that end, our approach of following the efficacy of incisional wound healing using TEWL measurements with time is well-suited to address temporal progress of healing including in different pathologies that influence tissue repair. For slower healing wounds, faster closure and effective tissue repair are imperative in order to prevent F I G U R E 3 Functional and biomechanical recovery of skin following suture closure and LASE sealing in Balb/c mice. (a) Photothermal response of LASE-skin interface during in vivo sealing irradiated using a continuous wave NIR laser tuned to 808 nm at a laser power density of 5.1 W/cm 2 . The region shaded in light blue color (temperature range from~50 C to~60 C) indicates the optimal temperature window for laser tissue sealing. The photothermal response curve shows data that are a mean of n = 3 independent experiments. (b) Representative images of 1-cm long skin incisions closed with four, simple interrupted 4-0 nylon sutures or LASE on Days 0 (immediately after closure), 2, 4, and 7 postwounding; control is unwounded skin surgically prepared similarly to incised skin. (c) Representative image showing three approximate locations at which TEWL measurements were carried out (white arrows) for each type of closure method. (d) Transepidermal water loss (TEWL) of healed skin and unwounded control skin on Days 2, 4, and 7 postwounding. TEWL value (in g/m 2 h) for each incision type is the average TEWL measurement from three nonoverlapping spots over the incision line shown in b. Data shown are mean ± standard error of the mean of n = 6 mice. (e) Ultimate tensile strength (UTS) and recovery, that is, %UTS of intact skin strength (secondary axis shown in red) of healed skin on Days 2 and 7 postwounding for suture-closed and LASE-sealed incisions. Data shown are mean ± standard error of the mean of n = 6 mice. Statistical significance was determined using two-way ANOVA followed by Fisher's LSD test and individual p values are shown; p < 0.05 are considered statistically significant.

| In vivo sealing of skin incisions: Barrier function recovery and healed skin strength
infections and further complications. To that end, the LASE approach, which shows better barrier function (TEWL) and biomechanical (UTS) recovery at earlier time points (Day 2), has the potential to also engender better outcomes in hosts with slower healing wounds.

| US and PAI
For PAI, a full spectrum scan between wavelengths of 680-980 nm, with a 5-nm increment, was carried out for a representative center slice of every skin sample. LASE PA signal shows a maximum at 800 nm, which is expected given the absorption of ICG. Control skin displays weak PA signal across all wavelengths, while the black-colored suture produces strong broadband signal. For PAI and spectral unmixing, signal data must be acquired at minimum of three wavelengths to distinguish three separate constituents (i.e., skin, suture, LASE). Observing the PA signal spectrum of each constituent, wavelengths of 680, 800, and 960 nm are chosen for the spectral unmixing. A 3D scan is then carried out for each sample with data being acquired at the three unmixing wavelengths (Figure 4a-c). It was qualitatively observed that there was a considerable drop in normalized PA signal at 800 nm on Day 7 postclosure compared to Day 2 postclosure (Figure 4b,c). This is further observed as reduced PA signal from

| Histological evaluation of LASE-sealed and suture-closed skin sections
During wound healing, re-epithelialization is a crucial step for restoring barrier function and preventing exposure to pathogens that cause surgical site infections. 37 We visualized the cellular and tissue processes that lead to skin healing using a histological analysis (Figure 6a We also evaluated the histological scar area, observed through picrosirius staining, to determine if there is a difference in initial scarring during the healing period in incisions closed with suture or LASE (Figure 6b,e). 38 Histological scar areas of 0.07 ± 0.01 and 0.08 F I G U R E 4 Normalized sample PA signal. Computed transverse slices of Bmode scans co-registered with normalized PA signal at 680, 800, and 960 nm for (a) control skin without any incision surgically prepped similarly to skin samples with incisions (b) skin incisions sealed using LASE at Day 2 postclosure and sealing (c) skin incisions sealed using LASE at Day 7 postclosure and sealing. Co-registered B-Mode and PA images are obtained by selecting a slice from the 3D scan data set that corresponds with approximately 500 μm subsurface depth (scale bar in yellow = 5 mm). Neutropenia is associated with slower healing and deficiency of several signals involved in neutrophil function can result in impaired healing. 40

| US evaluation of in vivo sealed incisions in live mice
We sought to evaluate the fidelity of US to interrogate LASE-and suture-sealed incisions in live mice using a portable, clinical US system; the portable nature and clinical application of this system was considered useful for potential translational applications. Live, anesthetized mice were evaluated with a linear probe transducer in B-mode operating at 22 MHz. Manually collected US images were compared to matched H&E-stained sections from the same mice and the linear dimension of the wound width was compared ( Figure 8).
We found a high degree of correlation between US and histopathology measurements (linear regression y = 1.140x À 182.5; R 2 = 0.985, N = 4 each group). Thus, in vivo US, using a clinically relevant system, provides an accurate representation of wound properties in mice with incisions sealed by both sutures and LASE.

| CONCLUSION
Tissue adhesives are an alternative and effective method of skin closure following surgical incisions or traumatic lacerations. Here, we comprehensively evaluated the efficacy and quality of silk fibroin-ICG based LASE for rapid sealing of skin incisional wounds in mice compared to conventional suturing using a temporal study of functional, biomechanical, and histological evaluation in addition to US and PAI. However, it is important to note that mouse skin heals by contraction, which poses significant limitations in using mouse models as indicators of scarring. Further studies in relevant animal models (e.g., porcine models) will be key to further compare scarring caused by sutures and LASEs.
Evaluation of translational potential of LASEs for application in humans will also require detailed studies in porcine models, studies in animal models of specific pathologies including slow-healing wounds (e.g., in diabetes) and wounds that are susceptible to infection. To that end, future work will involve a comprehensive investigation into bioactives that can accelerate tissue repair following laser sealing and into delivery of effective antimicrobial drugs for combating infections. In all these studies, a comprehensive picture of functional, biomechanical, and histological performance of LASEs will be obtained in order to investigate the potential for translating this technology for clinical use.